Identification of clinically relevant cancer chemoprevention targets has been challenging. Our multidisciplinary team (Precision Liver Cancer Prevention Consortium) will employ an innovative reverse-engineering approach, starting from transcriptome analysis of archived clinical specimens with long-term clinical follow-up, then moving to multifold experimental verification of only clinically well-validated targets, to elucidate chemoprevention targets with the highest likelihood of successful clinical application. With this approach, we could successfully identify liver cancer risk signatures and chemoprevention dugs, leading to a clinical trial (NCT02273362). To achieve our long-term goal of establishing clinically applicable chemoprevention strategies, here we aim to elucidate molecular dysregulation underlying carcinogenic milieu in livers affected with non- alcoholic steatohepatitis (NASH), the fastest rising liver cancer etiology, as clues to refined chemoprevention targets, drugs, and biomarker assays, which are expected to enable personalized patient management and more cost-effective liver cancer chemoprevention clinical trials, and lead to revolutionary improvement of patient prognosis and establishment of a new paradigm, reverse-engineering precision cancer prevention. Aim 1. Computationally-targeted screening of liver cancer chemoprevention agents. Candidate liver cancer chemopreventive compounds will be computationally prioritized, and screened together with LPA pathway inhibitor library in liver cancer risk signature-inducible cell system for the gene signature reversal. For selected compounds in the screen, mechanisms of action will be interrogated by gain- or loss-of-function assessment in the cell system. Aim 2. Functional validation of candidate liver cancer chemoprevention agents. In vivo liver cancer chemopreventive effect of the candidate agents will be validated in a diet-induced fibrotic/carcinogenic rat model mimicking global human cirrhosis transcriptome. Human relevance of the agents will be evaluated in organotypic ex vivo culture of clinical fibrotic liver tissues (n=30). To determine target cell type(s) and mechanisms of action for the agents, major hepatic cell types will be isolated from the rats, and transcriptome profiling will be performed to assess liver cancer risk signature member genes, the inferred target genes for the compounds, and related molecular pathways. Human fibrotic/cirrhotic NASH livers will be similarly profiled for cell type-specific transcriptomic dysregulation to verify that the modulated genes are relevant in human. Aim 3. Development of tissue- and serum-based liver cancer risk biomarker assays. Liver tissue-based cancer risk signatures will be implemented in clinically applicable tissue- (NanoString) and serum- (Luminex) based assays, and evaluated for technical validity and capability to predict future cancer risk in a cohort of 200 NASH patients with paired serum and liver tissue specimens as well as completed long-term clinical observation for cancer development.